title: Characterization of the two-step pathway for inhibition of thrombin by alpha-ketoamide transition state analogs.
abstract: The interaction of thrombin with several potent and selective alpha-ketoamide transition state analogs was characterized. L-370, 518 (H-N-Me-D-Phe-Pro-t-4-aminocyclohexylglycyl N-methylcarboxamide) a potent (Ki = 90 pM) and selective (>10(4)-fold versus trypsin) ketoamide thrombin inhibitor was shown to bind thrombin via a two-step reaction wherein the initially formed thrombin-inhibitor complex (EI1) rearranges to a more stable, final complex (EI2). A novel sequential stopped-flow analysis showed that k-1, the rate constant for dissociation of EI1, was comparable to k2, the rate constant for conversion of EI1 to EI2 (0.049 and 0.035 s-1, respectively) indicating that formation of the initial complex EI1 is partially rate controlling. Replacement of the N-terminal methylamino group in L-370,518 with a hydrogen (L-372,051) resulted in a 44-fold loss in potency (Ki = 4 nM) largely due to an increase in k-1. Consequently in the reaction of L-372,051 with thrombin formation of EI1 was not rate controlling. Replacement of the P1' N-methylcarboxamide group of L-370,518 with an azetidylcarboxamido (L-372,228) produced a 58-fold increase in the value of the equilibrium constant (K-1) for dissociation of EI1. Nevertheless, L-372,228 was a 2-fold more potent thrombin inhibitor (Ki = 40 pM) than L-370,518 due to its 16-fold higher k2 and 10-fold lower k-2 values. The desketoamide analogs of L-370,518 and L-372,051, namely L-371,912 and L-372,011, inhibited thrombin via a one-step process. The Ki value for L-371,912 and the K-1 value for its alpha-ketoamide analog, L-370,518, were similar (5 and 14 nM, respectively). Likewise, the Ki value for L-372,011 and the K-1 value for its alpha-ketoamide analog, L-372,051, were similar (330 and 285 nM, respectively). These observations are consistent with the view that the alpha-ketoamides L-370,518 and L-372,051 form initial complexes with thrombin that are similar to the complexes formed by their desketoamide analogs, and in a second step the alpha-ketoamides react with the active site serine residue of thrombin to form a more stable hemiketal adduct.
authors.affiliation: Department of Biological Chemistry, Merck Research Laboratories, West Point, Pennsylvania 19486, USA.
authors.forename: S D
authors.initials: SD
authors.lastname: Lewis
authors.forename: B J
authors.initials: BJ
authors.lastname: Lucas
authors.forename: S F
authors.initials: SF
authors.lastname: Brady
authors.forename: J T
authors.initials: JT
authors.lastname: Sisko
authors.forename: K J
authors.initials: KJ
authors.lastname: Cutrona
authors.forename: P E
authors.initials: PE
authors.lastname: Sanderson
authors.forename: R M
authors.initials: RM
authors.lastname: Freidinger
authors.forename: S S
authors.initials: SS
authors.lastname: Mao
authors.forename: S J
authors.initials: SJ
authors.lastname: Gardell
authors.forename: J A
authors.initials: JA
authors.lastname: Shafer
mesh_terms: D001665:Binding Sites; D001667:Binding, Competitive; D002384:Catalysis; D017022:Flow Injection Analysis; D005456:Fluorescent Dyes; D007700:Kinetics; D008956:Models, Chemical; D009842:Oligopeptides / Q000494:pharmacology; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D013917:Thrombin / Q000037:antagonists & inhibitors*
chemical_list: D005456:Fluorescent Dyes; D009842:Oligopeptides; D015842:Serine Proteinase Inhibitors; D013917:Thrombin